JP4654332B2 - New Yarrowia yeast and biological treatment method of waste water - Google Patents

Description

  The present invention relates to a novel yeast belonging to the genus Yarrowia and biological treatment of wastewater, and more particularly, the property of symbiosis with bacteria and assimilating animal and plant fats and oils at a high rate under strongly acidic to strongly basic conditions The present invention relates to a novel Yarrowia genus yeast having sewage and a biological treatment method of wastewater that reduces fats and oils in wastewater by introducing the cultured yeast.

  It is known that wastewater from recent food manufacturing factories and restaurants contains a lot of fats and oils. For example, dairy factories producing ice cream and fresh cream contain a large amount of milk fat, soy and other food processing factories contain vegetable oils, and restaurant effluents contain vegetable oils and animal fats. include. Wastewater with a high fat content becomes difficult to be treated with microorganisms, and it is necessary to physically and chemically remove the fat from the wastewater. That is, as a method for treating such fat-containing wastewater, n-hexane extract is used. As for 200-500mg / L, install and process oil and fat separation tank, and when it exceeds 500mg / L, install a pressurized flotation separation tank to remove oil and fat and lower n-hexane extract. Is desired. When these are released into public water areas, it is necessary to reduce the oil content below the regulation value by performing treatments such as activated sludge treatment and contact oxidation treatment. As a result, the wastewater treatment process itself is complicated, and the equipment costs are increased, which makes it difficult for wastewater containing a large amount of fats and oils.

Then, the method (refer patent documents 1 and 2) of assimilating and decomposing animal and vegetable fats and oils using various fats and oils assimilating yeast is proposed. Moreover, the method (refer patent document 3) of fixing the fat-and-oil assimilation yeast to a contact material or making it adhere, and biologically processing the waste_water | drain containing fats and oils is known.
Patent No. 2951683 Patent No. 2813265 JP-A-5-220493

  In the technical field where it is desired to reduce fats and oils in oil-containing fat wastewater in a wide pH range discharged from each manufacturing company, various business establishments and households, the present invention is used to assimilate the fats and oils in wastewater. Although it has been studied that it is effective, the conventional technology requires special equipment and equipment such as attaching yeast to the yeast treatment tank and contact material, and the activity decreases when bacteria are mixed in, so the yeast alone Or, the deterioration of the equipment is greatly promoted by adding and adjusting the acidifying agent, basifying agent, or chlorinating agent in the wastewater to make the optimal growth environment where yeasts grow. It is a problem. Production facilities such as food products are gently drained from low pH (pH) to high pH by using acidic cleaners or basic cleaners for stationary cleaning, removing oils and fats at a high rate and changing pH Yeasts that are strong and can coexist with various bacteria are desired. In addition, there is a need for an environmentally friendly wastewater biological treatment method that does not use chemical substances, etc., and provides a satisfactory yeast that has an improvement effect on these, and uses this cultured yeast to remove fats and oils in the wastewater. An object of the present invention is to provide a biological treatment method for reducing the biological treatment.

  The present inventors have focused on biological treatments that assimilate fats and oils under a wide range of pH conditions that do not promote the deterioration of facilities as described above and enable environmentally friendly treatments. The present inventors have found that there are yeasts belonging to the genus Yarrowia that specifically coexist with bacteria and that efficiently utilize animal and plant oils and fats with strongly acidic to strongly basic wastewater.

  Accordingly, the present invention provides a yeast belonging to the genus Yarrowia that is symbiotic with bacteria and highly assimilate animal and vegetable oils under conditions of strong acidity to strong basicity, and the yeast is administered to an oil-containing fat wastewater. The present invention provides a biological treatment method for wastewater characterized by

Yeast belonging to the genus Yarrowia that highly assimilate animal and vegetable oils and fats under strongly acidic to strongly basic conditions can be separated and collected from wastewater or soil by, for example, the screening method described below. It has been studied that yeast belonging to the genus Yarrowia can assimilate petroleum and use protein, and has an advantage that it can be used for purification of a wide range of wastewater and yeast can be used as feed. In addition, in this specification, utilization of fats and oils or reducing fats and oils means reducing n-hexane extract. In addition, symbiosis means that the number of yeasts is 1 × 10 ⁶ cells / ml or more even if other bacteria are present, strongly acidic means pH 3 or less, and strongly basic means pH 10 or more. Saying that high utilization of fats and oils means that about 1,000 mg of fats and oils in one liter is reduced by 90% or more.

  As a screening method for the yeast, yeast generally prefers neutral to acidic. However, the yeast can be adjusted to pH 9 to 10 and can be grown basic, and can coexist with bacteria. An antibacterial agent is not used in primary culture to select yeast, but is used in plates during pure separation. For example, yeast is separated from wastewater and soil from a food manufacturing company. First, a culture solution for screening yeast that assimilate fats and oils is prepared. 5.0 g of ammonium sulfate and hydrogen phosphate are added to ion-exchanged water. Dissolve 0.5g of dipotassium, 0.25g of magnesium sulfate heptahydrate, 5.0g of calcium carbonate, make 1 liter and sterilize at 121 ° C for 20 minutes, and then sterilize so that the fat content is about 1,000 mg / L A culture solution containing added commercial milk or sterilized commercial soy milk is placed in a Sakaguchi flask and cultured with shaking at an appropriate temperature. One platinum loop of the above culture solution is taken and streaked on a Sabouraud medium to which an appropriate amount of antibiotics has been added. The emerged colony is taken and cultured again using the culture solution adjusted to pH 3 to 4, and the culture solution is symbiotic with bacteria by taking one platinum loop and separating and cultivating the fats and oils from strong acidity to strong basicity. Obtain yeast to assimilate. From here, yeast of the genus Yarrowia can be selected by DNA analysis.

  A general activated sludge method can be applied to this treatment method. First, the wastewater is led to the adjustment tank, where the pH is adjusted so that it is suitable for the growth of activated sludge microorganisms, and the nutrient concentration is adjusted. The adjusted wastewater is sent to the aeration tank, and oxygen is sufficiently supplied. Then, the sludge microorganisms are activated to decompose organic matter, the treatment liquid is stored in a sedimentation tank, the sludge is settled, and the supernatant is separated and discharged. Part of the sludge that has settled and separated is sent back to the aeration tank for reuse as sludge. In this process, the adjustment tank and the aeration tank are suitable for the place where Yarrowia yeast is introduced. Moreover, if it is a contact aeration method, an adjustment tank and a contact aeration tank are suitable similarly. In addition, when using Yarrowia genus yeast, adjustment of pH of a control tank is not required if it is less than 1.8-10.0.

  Next, as one representative example selected from the above screening method, the mycological properties of Yarrowia sp. YH-01 yeast are shown.

DNA analysis YM agar (Becton Dickinson, MD, USA) Genomic DNA was extracted from specimens cultured at 25 ° C for 1 week (using FastPrep FP120 and Fast DNA Kit), and similar nucleotide sequences were obtained from the international DNA database (GenBank). / EMBL / DDBJ), a homology search by BLAST was performed. As a result, in DNA analysis (28 Sr DNA-D1 / D2), the base sequence of Yarrowia lipolytica species whose base sequence of Yarrowia YH-01 is a kind of Ascomycetous yeasts and 99. A homology rate of 6 to 100% was shown, and it was estimated to be the same species.

Culture Properties and Morphological Features YM agar (Becton Dickinson, MD, USA) plate medium was inoculated and cultured for 1 month from 25 ° C. for 1 month, and colony properties were observed with the naked eye and a stereomicroscope. The observation items of microscopic features were based on Barnett et al. (2000) and Kurtzman and Fell (1998).
(1) Wet and white colonies (2) Egg-shaped to oval vegetative cells (3-5 μ × 2-3.5 μ)
(3) Bipolar or multipolar budding (4) No formation of pseudohyphae (pseudohyphae and fungal hyphae) was observed.
(5) No formation of pupa which is a sexual reproductive organ.

Biochemical property test Barnett et al. (2000) and Kurtzman and Fell (1998), carbon source utilization test, nitrogen source utilization test, saccharide fermentation test, vitamin requirement test, temperature tolerance test, drug resistance Tables 1 to 5 show the results of the tests and the biochemical property tests for the pH tolerance test and the oil and fat assimilation test. “+” In the table indicates a positive reaction, “−” indicates a negative reaction, and “w” indicates a weak positive reaction. The culture temperature other than the temperature tolerance test was 25 ° C. Yarrowia YH-01 is YH-01, Yarrowia Lipolytica is Y. It is described as 1.

  In the DNA analysis (28 Sr DNA-D1 / D2) of yeast newly isolated from wastewater by the present inventors, the base sequence of Yarrowia YH-01 is 99.6 to 100% homologous to the base sequence of Yarrowia lipolytica. The rate was estimated and the same species. Since the genus Yarrowia is a genus, it was extracted from the data of Barnett et al. (2000) and compared with Yarrowia lipolytica. In terms of morphological and culture characteristics, formation of pseudohyphae (pseudohyphae and fungal hyphae) and formation of pupae which are sexual reproductive organs were not observed. In addition, the biochemical properties of Yarrowia lipolytica described in Barnett et al. (2001) are compared and shown in Tables 1 to 4, but both assimilate using glucose as a carbon source. It was shown that it was different from Yarrowia lipolytica in some items such as assimilability of nitrogen source, temperature tolerance and drug resistance.

Considering these points, in the comparison of mycological properties, Yarrowia YH-01 could not be identified as Yarrowia lipolytica , and was considered a new species or a subspecies. In addition, as shown in Table 5, the yeast grew under strong acidity to strong basic conditions, assimilated the fats and oils during culture, and showed excellent utility in reducing n-hexane extract. Based on the taxonomic position of this fungus is Kirk et al. (2001), Ascomycota, Ascomycetes network, Saccharomyces Amo, Saccharomyces Mike scan eyes, Jipodoakusu family, as a novel microorganisms in the new species or subspecies of Yarrowia The yeast was deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology on July 6, 2004, and is stored under the deposit number FERM P-20114 .

  Moreover, in the biological treatment method of waste water using the novel Yarrowia genus yeast according to the present invention, it is preferable to introduce Yarrowia YH-01.

  Yarrowia yeast that assimilate the animal and vegetable oils and fats of the present invention at a high rate is symbiotic with bacteria by putting them in oil-containing fat drainage, and the outstanding effect of reducing fats and oils under strongly acidic to strongly basic pH conditions It plays. Therefore, wastewater from food manufacturing plants does not require the addition of acids, basifying agents, and chlorinating agents to control bacteria to adjust the wastewater to the optimum pH of the microorganisms used. It solves the problem of deterioration and plays an important role in improving the environment widely for reducing fats and oils as a safe biological treatment method for fats and oils without using chemical substances. It can be added directly to an existing adjustment tank, activated sludge tank or contact oxidation tank. Moreover, since it can grow from strong acidity to strong basicity, the enzyme extracted from this bacterium is expected to have activity under a wide range of pH conditions, and can be applied to a wide range of applications other than wastewater treatment.

  Next, the present invention will be described with reference to examples, but test examples using cultured yeasts have been shown, but the present invention is not limited to these examples. In the following examples, “part” and “%” are based on weight unless otherwise specified.

Yeast selection example

  To select yeasts that assimilate fats and oils from strongly acidic to strongly basic, 5.0 g of ammonium sulfate, 0.5 g of dipotassium hydrogen phosphate, 0.25 g of magnesium sulfate heptahydrate, calcium carbonate in ion-exchanged water Dissolve 5.0 g to 1 liter (culture medium A), sterilize at 121 ° C. for 20 minutes, and add sterilized commercial milk or sterilized commercial soy milk so that the fat content is about 1,000 mg / L The obtained culture solution is adjusted to pH 10 with caustic soda solution, and cultured with shaking in a Sakaguchi flask at 27 ° C. for 5 days. One platinum loop of the above culture broth was streaked on Sabouraud medium (Eiken Chemical Co., Ltd.) supplemented with chloramphenicol (75 mg / L) and cyclohexamide (50 mg / L) and cultured at 27 ° C. for 3 to 5 days. To do. The emerged colony was picked, the culture solution was adjusted to pH 3 with hydrochloric acid, cultured in the same manner, the culture solution was picked up from one platinum loop, streaked on a Sabouraud medium, cultured at 27 ° C. for 3 to 5 days, and appeared. The colonies were dispersed in sterile physiological saline and streaked again on the Sabouraud medium to screen as yeasts that assimilate fats and oils from strong acidity to strong basicity. As a result of selection of about 200 kinds of waste water and soil by the present inventor, yeasts that assimilate 90% or more of milk fat and soybean fat are selected, and three Yarrowia yeasts are obtained from these yeasts. YH-01, YH-02, and YH-03 were used.

Example of cultured yeast production

Y. Sterilized commercially available milk or sterilized so that the amount of fat is about 1,000 mg / L in the sterilized culture solution A by aseptically taking the preserved yeast Yarrowia YH-01 grown on M agar medium (Difco YM Ager) The inoculated culture solution was added to the commercial soymilk, and cultured with aeration and agitation at 27 ° C. for 48 to 72 hours. The culture broth thus obtained was used as Yarrowia YH-01 culture yeast, and this was freeze-dried according to a conventional method to obtain freeze-dried yeast. The number of bacteria contained in this cultured yeast was 10 ⁷ to 10 ⁸ / ml, and the number of bacteria contained in freeze-dried yeast was 10 ⁸ to 10 ⁹ / g.

Sterilized commercially available milk or sterilized commercial soymilk is added to the sterilized culture solution A so that the fat amount is about 1,000 mg / L, and the pH is adjusted to 2.0, 4.0, 6 with hydrochloric acid and caustic soda solution. 0.0%, 8.0, 10.0, 3% viable yeast cells (7.3 × 10 ⁷ / ml) cultivated in the same manner as in the production example in advance and added at 3% at 27 ° C. Cultured for days. The results are shown in Table 6. It was revealed that milk fat and soybean oil were assimilated at a high rate in the acidic range, neutral range, and alkaline range, and the n-hexane extract was greatly reduced.

As an acidic drainage model in which bacteria coexist, a sterilized culture solution A is prepared and added to a commercial fermented milk containing live lactic acid bacteria so that the milk fat content is 1,000 mg / L. The number of lactic acid bacteria; 2.4 × 10 ⁶ / ml) was adjusted to pH 5.0, and the live cells of Yarrowia YH-01 previously cultured in the same manner as in the production example (7.6 × 10 ⁷ / ml) 3% was inoculated and cultured at 27 ° C. for 5 days. The results are shown in Table 7. It was revealed that the fats and oils were sufficiently assimilated even in the presence of lactic acid bacteria, and the n-hexane extract was greatly reduced. Moreover, the number of bacteria of Yarrowia YH-01 on the 5th day was 6.6 × 10 ⁷ / ml, and it became clear that oil and fat decreased under acidic conditions and symbiosis with bacteria.

As a basic drainage model in which bacteria are present, a sterilized culture solution A similar to that in Example 2 was prepared, and sterilized commercial soymilk was added so that the soymilk fat content was 1,000 mg / L. Viable cells of Yarrowia YH-01, which was adjusted to pH 9.0 and cultured in the same manner as in the production example in a test solution (Bacterial subtilis; 5.3 × 10 ⁵ / ml) to which Bacillus subtilis was added. (7.6 × 10 ⁷ / ml) was inoculated with 3% and cultured at 27 ° C. for 5 days. The results are shown in Table 8, and it was revealed that soybean oil and fats were sufficiently assimilated even if other bacteria were present in basicity, and the n-hexane extract was greatly reduced. In addition, the number of bacteria on the fifth day of Yarrowia YH-01 was 5.4 × 10 ⁷ / ml, and it became clear that the fat and oil decreased under basic conditions and coexisted with bacteria.

Drainage of n-hexane (1,100 mg / L), BOD (850 mg / L), pH 8.3 was collected from a wastewater adjustment tank of a milk manufacturing factory, and freeze-dried powder Yarrowia YH prepared in the same manner as in the production example. An example in which 0.3% of −01 (6.6 × 10 ⁸ / g) was inoculated is shown. Here, as a model in which Yarrowia YH-01 is added to an adjustment tank and an activated sludge tank of a general activated sludge method, the results of aeration and agitation culture at 27 ° C. for 3 days are shown. As shown in Table 9, in the drainage model in which various bacteria are present, the reduction rate of n-hexane in the drainage on the third day decreased the fat and oil content by 90% or more, and the third day of Yarrowia YH-01 The number of bacteria was 5.2 × 10 ⁷ / ml, and it became clear that the fats and oils decreased in the actual waste water and symbiotic with bacteria. Further, after sterilizing and cooling the waste water at 95 ° C., 0.3% of freeze-dried powder of Yarrowia YH-01 was added and cultured in the same manner. As a result, no significant difference was observed.

Claims (3)

Yeast belonging to the genus Yarrowia that symbiotic with bacteria and assimilate at least 90% of 1 ppm of animal and vegetable fats and oils under strongly acidic to strongly basic conditions. The yeast according to claim 1, which is Yarrowia YH-01 (FERM P 20114). A biological treatment method of wastewater, characterized in that fats and oils are reduced by introducing the cultured yeast according to claim 1 or 2.